Structural Investigations of the Nickel-Induced Inhibition of Truncated Constructs of the JMJD2 Family of Histone Demethylases Using X-ray Absorption Spectroscopy

Abstract

Occupational and/or environmental exposure to nickel has been implicated in various types of cancer, and in vitro exposure to nickel compounds results in accumulation of Ni(II) ions in cells. One of the major targets of Ni(II) ions inside the cell is Fe(II)- and αKG-dependent dioxygenases. Using JMJD2A and JMJD2C as examples, we show that JMJD2 family of histone demethylases, which are products of putative oncogenes as well as Fe(II)- and αKG-dependent dioxygenases, are highly sensitive to inhibition by Ni(II) ions. In this work, X-ray absorption spectroscopy (XAS) has been used to investigate the Fe(II) active site of truncated JMJD2A and JMJD2C (1 – 350 aa) in the presence and absence of αKG and/or substrate to obtain mechanistic details of the early steps in catalysis that precede O2 binding in histone demethylation by the JMJD2 family of histone demethylases. Zinc K-edge XAS has been performed on the resting JMJD2A (with iron in the active site) to confirm the presence of the expected structural zinc site. XAS of the Ni(II)-substituted enzymes has also been performed to investigate the inhibition of these enzymes by Ni(II) ions. Our XAS results indicate that the five-coordinate Fe(II) center in the resting enzyme is retained in the binary and ternary complexes. In contrast, the Ni(II) center is six-coordinate in the resting enzyme, binary and ternary complexes. XAS results indicate that both Fe(II) and Ni(II) bind αKG in the binary and ternary complexes. The electron density build-up that is observed at the Fe(II) center in the presence of αKG and substrate is not observed at the Ni(II) center. Thus, both electronic and steric factors are responsible for Ni-induced inhibition of the JMJD2 family of histone demethylases. Ni-induced inhibition of these enzymes may explain the alteration of the epigenetic mechanism of gene expression that is responsible for Ni-induced carcinogenesis.

Epigenetic dysregulation by nickel through repressive chromatin domain disruption

“…Previous studies have shown that the JmjC domain H3K9me2 demethylases could be inhibited by Ni, resulting in an increase in the total levels of H3K9me2 (19,36,37). Taken together, our results suggest that, although depletion of CTCF could initiate spreading of H3K9me2 and gene silencing, the effect is more robust in Ni-exposed cells, where reduction in CTCF binding could be accompanied by a reduction in demethylase activity, thus augmenting H3K9me2 spreading and gene silencing (Fig.…”

“…The demethylation of lysines by JHDMs occurs by catalyzing the generation of oxidized iron. Ni inhibits this family of enzymes by displacing the iron (19,36,37). Previously, loss of H3K9 demethylase LSD1 has been demonstrated to result in spreading of H3K9me2 (51).…”

10th NTES Conference: Nickel and arsenic compounds alter the epigenome of peripheral blood mononuclear cells

“…Once in the cell, nickel can affect histone modifications by inhibiting dioxygenases [19] or histone acetyltransferases (HATs). Nickel's potential to induce gene silencing is mediated by increased condensation of the genomic region followed by DNA methylation both of which facilitate heterochromatinization [20].…”

“…Nickel has been shown to displace iron in the active site of these enzymes, which inhibits their enzymatic function [19]. A study by Chen et al 2010 found that nickel binds to the iron binding motif in dioxygenases with three times more affinity than iron [21].…”

The Jumonji family: past, present and future of histone demethylases in cancer

“…KDM4 family inhibitors will likely require a much more detailed chemical and biochemical analysis and longer preclinical trials. Although several chemically different inhibitors have been proposed, X-ray crystal structures of at least some Jumonji targets have demonstrated an indirect effect, likely mediated by alteration of complex composition (41,(109)(110)(111). The availability of other X-ray crystal structures will represent a crucial step in the development of novel Jumonji modulators.…”